Retained placenta (RP), a condition that fetal membranes failed to expel within 24 h of calving, is one of the most prevalent and costly diseases in dairy cows. Our objective was to discover molecular biomarkers and pathways, which can aid in screening, prediction and/or early diagnosis of RP. Using a nested case-control design, we collected 21, 14, 7 d before calving and directly after calving serum samples in healthy dairy cows that subsequently did (RP; n = 8) or did not (Control; n = 9) develop RP. After ethanol extraction, samples were injected onto an Acquity amide column (Waters Corporation) coupled to the SYNAPT G2 mass spectrometer. Feature annotations and structural elucidation were assigned using an in-house library and online databases (METLIN, HMDB, LIPIDMAPS), and matching MS/MS and MS^E data. Data analysis was conducted in PROC MIXED of SAS version 9.4. Fixed effects were collection time, group, and their interaction. Repeated measures within cows were modeled using a first-order heterogeneous variance-covariance matrix. Group differences were most pronounced 7 d before calving and showed significant differences in 44 of 81 annotated metabolites; 9 carnitines, 23 amino acids and their metabolites, 3 conjugated bile acids, 2 carbohydrates, and 3 phospholipid precursors were all higher in RP-vs. Control cows. Complete group separation was achieved by 1 [methyl ethanolamine phosphate (MEP)], 3 (free carnitine, choline, MEP), 9 (free carnitine, Car C4:0, -C16:0, -C18:0, MEP, trimethyl-lysine, histidine, phenylalanine, taurine), and 1 metabolite (choline) at 21, 14, 7, and 0 d before calving, respectively (all higher in RP vs. Control cows). The best early RP indicator was MEP (signal intensities were 2.3-fold, 2.7-fold, 5.4-fold, and 2.3-fold higher in RP- vs. Control cows at 21, 14, 7, before and directly after calving, respectively. In conclusion, LC-MS metabolomic serum signatures indicate that global challenges in protein, phospholipid, and fatty acid metabolism precede RP in transition dairy cows.